Method of producing microbeads

ABSTRACT

A method for producing microbeads comprising an active component encapsulated within a gelled polymer matrix comprises the steps of providing a suspension of denatured whey protein and an active component, treating the suspension to generate microbeads, and immediately curing the microbeads by acidification. The microbeads are discrete droplets of gelled whey protein having an average diameter in the micron range (for example, from 80 to 500 microns) and which, suitably have a generally spherical shape. The microbeads are capable of surviving passage through the stomach, and delivering the encapsulated active agent in the instestine. Ex-vivo and in-vivo data shows that active agent encapsulated within microbeads retains its functionality upon delivery to the intestine, and that coating of the microcapsules allows targeted delivery of the active agent to the distal part of the intestine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/EP2010/054846 filed Apr. 13, 2010, which claims the priority of U.S.Provisional Application No. 61/168,746, filed on Apr. 13, 2009. Thecontents of both applications are hereby incorporated by reference intheir entirety.

INTRODUCTION

The invention relates to a method for producing microbeads comprising anactive component encapsulated within a gelled polymer matrix. Inparticular, the invention relates to a method for producing microbeadscomprising sensitive components such as probiotic bacteria encapsulatedwithin a gelled polymer matrix. The invention also relates to microbeadscomprising an active component encapsulated within a polymer matrix.

BACKGROUND TO THE INVENTION

The method of using whey proteins for creating whey protein gel-beads isnot novel. Ainsley Reid et al., 2005 (Ainsley Reid, A., J. C.Vuillemard, M. Britten, Y. Arcand, E. Farnworth, and C. P. Champagne.2005. Microentrapment of probiotic bacteria in a Ca (²⁺)-induced wheyprotein gel and effects on their viability in a dynamicgastro-intestinal model. Journal of Microencapsulation 22:603-19.)described a method that involves the production of simple droplet beadsusing pipettes/syringes followed by a gelation step. This methodproduces very large bead gels that are mostly unsuitable for foodpreparation.

Rosenberg, 1994 (Rosenberg, M. 1993. Whey proteins as microencapsulatingagents—Microencapsulation of anhydrous milkfat—Structure evaluation FoodStructure 12:31. or Rosenberg, M. a. L., S. L. 1993. Microstructure ofwhey protein/anhydrous milkfat emulsions. Food Structure 12:267-274)described the emulsification of denatured whey protein mixture by highpressure homogenisation or high shear, followed by internal gelation andsubsequent separation of gel and oil phase. The major disadvantages ofthis method is that use of high pressure/high shear necessary for theformation of the micro-beads can cause significant damage to the activecomponent the micro-bead is encasing. Also, the presence of an oil phaseand its removal through the use of detergents can also be damaging tosome active components, like probiotic bacteria.

STATEMENTS OF INVENTION

The Applicant has surprisingly discovered that microbeads derived fromheat-denatured whey protein immediately gelled and cured in anacidification bath, form stable, robust and ideally spherical microbeadscapable of encapsulating sensitive components and protecting thecomponents from the low pH environment of the stomach, while also beingcapable of controlled release of the components in the more neutral pHconditions of the small intestine.

Accordingly, in a first aspect, the invention relates to a method forproducing microbeads comprising an active component encapsulated withina gelled polymer matrix, the method comprising the steps of providing asuspension of denatured whey protein and an active component, treatingthe suspension to generate microbeads, and immediately curing themicrobeads by acidification.

In this specification, the term “microbeads” should be understood tomean discrete droplets of gelled whey protein having an average diameterin the micron range (for example, from 80 to 500 microns) and whichsuitably have a generally spherical shape.

The pH of the acidification step should be close to the pI of theβ-lactoglobulin in the whey protein mixture. Preferably, theacidification step is carried out at a pH of from 4.0 to 4.9, morepreferably a pH of from 4.1 to 4.8, 4.2 to 4.7, 4.3 to 4.7, preferably apH of about 4.5 to 4.7, and ideally a pH of about 4.6.

Ideally, the whey protein is a whey protein isolate. However, othertypes of whey protein fractions may also be employed in the process ofthe invention, for example whey protein hydrolysate or whey proteinconcentrates.

Typically, the microbeads are cured in an acidification bath containingan acidic curing solution, suitably an acetate solution, although otheracids may be employed. The parameters of the acidification bath arechosen to ensure instantaneous gelation of the microbeads. That is tosay that the microbeads gel (i.e. harden) immediately upon contact withthe acid curing solution. It has been surprisingly found that if theparameters of the acidification bath are such that instantaneousgelation does not occur, the resultant micro-beads have irregular,non-homogenous, shapes. In contrast, when instantaneous gelation doesoccur, microbeads having a spherical, homogenous shape are produced. Theparameters required to achieve instantaneous gelation depend on thecharacteristics of the whey protein, including the degree ofdenaturation, and the type and amount of active component. Generally,the parameters of the acid curing solution that are varied are pH, acidconcentration, and temperature. In the example provided below, in whicha 9% WPI suspension having about 80% β-lactoglobulin (having a degree ofdenaturation of about 95%) is employed, and in which the activecomponent is a probiotic bacteria, the pH of the acidic solution is 4.2to 4.7, the acid concentration of 0.3 to 0.6 M, and the temperature ofthe acidic solution is from 30° C. to 40° C. Suitably, the pH of theacidic solution is 4.5 to 4.7, the acid concentration of 0.4 M to 0.6 M,and the temperature of the acidic solution is from 34° C. to 48° C.Ideally, the pH of the acidic solution is about 4.6, the acidconcentration is about 0.5 M, and the temperature of the acidic solutionif from 35° C. to 37° C.

Typically, the acidic curing solution comprises a surfactant to preventor inhibit agglomeration of the formed microbeads. Suitably, thesurfactant is a polysorbate surfactant, ideally Tween 20.

Suitably, the formed microbeads are subject to an extended curing periodin the acidification bath, for a period of at least 15 minutes (aftergelation), and preferably for a period of at least 20 minutes. In apreferred embodiment of the invention, the formed microbeads are curedfor a period of time from 20 to 180, 20 to 120, or 20 to 60 minutes.Ideally, the acidic curing solution is agitated during the curingprocess.

Generally, the method involves an initial step of denaturing the wheyprotein. Ideally, the whey protein is heat-denatured, although othermethods of denaturation are also applicable, for examplepressure-induced denaturation. In a preferred embodiment, the wheyprotein is heat-denatured at a temperature of from 75° C. to 80° C.,suitably for a period of between 30 minutes and 50 minutes. Typically,the whey protein is agitated during heat-denaturation.

Suitably, the concentration of the whey protein (prior to the additionof the sensitive component) is from 5 to 15%, preferably from 7 to 12%,and ideally from 9 to 11% (w/v). Typically, the denatured whey proteinis in the form of a soluble suspension (prior to addition of the activecomponent), in which partial gelation of the β-lactoglobulin hasproduced suspended protein agglomerates. Typically, the suspension issubject to a filtration process, generally prior to addition of theactive component. In one preferred embodiment, the suspension is passedthrough a plurality of filters having a gradually decreasing pore size.Ideally, the final filter has a sub-micron pore size, for example 0.1 to0.9 microns.

Ideally, the whey protein employed in the process of the invention has aβ-lactoglobulin content of at least 30%, 40%, 50%, 60%, 70% or 80%(w/w). Typically, the β-lactoglobulin has a degree of denaturation of atleast 60%, 70%, 80%, 90% or 95%.

Various methods will be apparent to the skilled person for generatingmicrobeads, for example prilling and spraying. A preferred method ofproducing the microbeads is a vibrating nozzle technique, in which thesuspension is sprayed through a nozzle and laminar break-up of thesprayed jet is induced by applying a sinusoidal frequency with definedamplitude to the spray nozzle. Examples of vibrating nozzle machines arethe ENCAPSULATOR (Inotech, Switzerland) and a machine produced by NiscoEngineering AG, or equivalent scale-up version such as those produced byBrace GmbH or Capsulæ and the like.

Typically, the spray nozzle has an aperture of between 100 and 600microns, preferably between 100 and 200 microns, suitably 140 and 160microns, and ideally about 150 microns.

Suitably, the frequency of operation of the vibrating nozzle is from 900to 3000 Hz. Generally, the electrostatic potential between nozzle andacidification bath is 0.85 to 1.3 V. Suitably, the amplitude is from 4.7kV to 7 kV. Typically, the falling distance (from the nozzle to theacidification bath) is less than 50 cm, preferably less than 40 cm,suitably between 20 and 40 cm, preferably between 25 and 35 cm, andideally about 30 cm. The flow rate of suspension (passing through thenozzle) is typically from 3.0 to 10 ml/min; an ideal flow rate isdependent upon the nozzle size utilized within the process.

In one embodiment, the process involves a step of detecting the size ofthe initial microbeads generated, comparing the detected size of themicrobeads with a predetermined desired size, and wherein the detectedsize differs significantly from the predetermined desired size, themicrobeads are diverted away from the acidification bath until thedetected size correlates with the predetermined desired size.

In one embodiment of the invention, the active component is a suspensionof cells, typically bacterial cells, ideally a probiotic bacteria cellsuspension. Suitably, the concentration of cells in the suspension isfrom 1×10⁷ to 5×10¹¹ cfu/ml, preferably from 1×10⁸ to 5×10¹⁰ cfu/ml,ideally from 1×10⁹ to 7×10¹⁰ cfu/ml.

In one aspect, the methods of the invention do not involve exposing themicrobeads to high pressure or high shear (i.e. homogenisation). Thisavoids damage to any components that are sensitive to pressure or shearsuch as, for example, prokaryotic or eukaryotic cells.

The invention also relates to a preparation of microbeads in which eachmicrobead (i.e. all or most of the microbeads) comprises an activecomponent dispersed within a gelled denatured whey protein matrix, andwherein at least 50%, 60%, 70%, 80% or 90% of the microbeads in thepreparation have a diameter in a 80 to 500μ range.

Typically, at least 90% of the microbeads in the preparation are capableof surviving intact in fresh ex-vivo porcine gastric juice of pH 2.0 forat least three hours at 37° C. under agitation at 150 rpm (employing themethods as described below).

Ideally, substantially all of the microbeads in the preparation arecapable of surviving intact in fresh ex-vivo porcine gastric juice of pH2.0 for at least three hours at 37° C. under agitation at 150 rpm(employing the methods as described below).

Typically, the whey protein matrix comprises at least 30%, 40%, 50%,60%, 70%, or 80% β-lactoglobulin (w/w). Suitably, the β-lactoglobulinhas a degree of denaturation of at least 50%, 60%, 70%, 80%, 90% or 95%.In a preferred embodiment of the invention, the whey protein comprisesat least 70% β-lactoglobulin having a degree of denaturation of at least90%.

Ideally, the whey protein is whey protein isolate (WPI) or whey proteinconcentrate (WPC).

Preferably, a majority of the microbeads in the preparation arespherical.

Suitably, the active component is homogenously dispersed within thepolymer matrix.

In a preferred embodiment, at least 90% of the microbeads in thepreparation have a diameter from 200 microns to 300 microns.

In a preferred embodiment of the invention, the active agent is a cell,preferably a probiotic cell preparation. Thus, the microbead preparationof the invention may be used to safely transport probiotic cells throughthe stomach and bile salt domain (where the cells are protected by themicrobeads from harsh gastric conditions) for delivery of cells to thedistal intestinal tract.

The invention also relates to a food product or beverage comprising amicrobead preparation of the invention. Various types of food productsare envisaged including dairy products (for example, yoghurt, milk,cheese, etc) and acidic fruit products.

The invention also relates to a vehicle capable of delivering an activeagent to a lower intestine of a subject while protecting the activeagent during passage through the stomach of the subject, the vehiclecomprising a microbead preparation according to the invention. Thus, asexplained above, the microbeads have a net positive charge and aresubstantially resistant to the low pH conditions of the stomach and, assuch, protect the encapsulated active agents entrapped within themicrobead. However, as the microbeads transit through the gut to theintestine, the pH of the environment increases to above the pI of thewhey protein, which causes the microbead gels to weaken, allowingendogenous proteolytic enzymes disintegrate the gel and release theentrapped active agents.

The invention also relates to a method of delivering an active agent toa lower intestine of a subject and protecting the active agent duringpassage through a stomach of the subject, and in which the active agentis susceptible to degradation in the acidic conditions of the stomach,the method comprising a step of orally administering a preparation ofmicrobeads according to the invention to the subject.

The invention also provides a method for producing coated microbeadscomprising the steps of producing microbeads according to a method ofthe invention, and re-suspending the microbeads in a solution of anionicpolysaccharide having a pH of 4.6 or less for a period of at least 10,30, 60, 90, 120, 180 minutes. In this specification, the term “anionicpolysaccharide” should be understood to mean a polysaccharide that has anet negative charge when dissolved or solubilised in a solution having apH of 4.6 or less. Examples of suitable anionic polysaccharides includepectins, alginates, carrageenans, and acacia gums (for example, ApplePectin, Citrus Pectin, Sodium Alginate, Kappa carrageenan, iotacarrageenan, and gum acacia).

Suitably, when the microbeads are coated with alginate, they are curedafter the coating step, for example, cured in a solution of calciumchloride (0.1-0.5 M, typically about 0.2 M).

The invention also relates to a preparation of coated microbeads inwhich each coated microbead (i.e. all or most of the coated microbeads)comprises a microbead coated with a layer of anionic polysaccharide, andwherein the microbead comprises an active component dispersed within agelled denatured whey protein matrix.

Typically, at least 50%, 60%, 70%, 80% or 90% of the microbeads in thepreparation have a diameter of from 80 to 500μ.

Suitably, at least 90% and ideally 100% of the coated microbeads in thepreparation are capable of (a) surviving intact in fresh ex-vivo porcinegastric juice of pH 1.8 for at least three hours at 37° C. underagitation at 150 rpm, and, optionally, (b) rupturing in fresh ex-vivoporcine jejunum juice of pH 6.59 within one hour at 37° C. underagitation at 150 rpm.

Suitably, at least 90% and ideally 100% of the coated microbeads in thepreparation are capable of (a) surviving intact in fresh ex-vivo porcinegastric juice of pH 1.8 for at least three hours at 37° C. underagitation at 150 rpm, and (b) rupturing in fresh ex-vivo porcineintestinal juice of pH 6.59 within 40 minutes at 37° C. under agitationat 150 rpm.

Generally, at least 90% or 100% of the coated microbeads in thepreparation are capable of surviving intact in fresh ex-vivo porcinegastric juice of pH 2.4 for at least two hours at 37° C. under agitationat 150 rpm, and wherein at least 50%, 60%, 70%, 80%, or 90% of thecoated microbeads rupture in fresh ex-vivo porcine jejunum juice of pH6.59 within 40 minutes at 37° C. under agitation at 150 rpm.

Typically, the anionic polysaccharide is selected from the groupconsisting of: pectin; alginate; carrageenan; and acacia. Preferably,the anionic polysaccharide is selected from the group consisting ofApple Pectin, Citrus Pectin, Sodium Alginate, Kappa carrageenan, iotacarrageenan, and gum acacia.

Typically, the whey protein matrix comprises at least 30%, 40%, 50%,60%, 70%, or 80% β-lactoglobulin (w/w). Suitably, the β-lactoglobulinhas a degree of denaturation of at least 50%, 60%, 70%, 80%, 90% or 95%.In a preferred embodiment of the invention, the whey protein comprisesat least 70% β-lactoglobulin having a degree of denaturation of at least90%.

Ideally, the whey protein is whey protein isolate (WPI).

Preferably, a majority of the coated microbeads in the preparation arespherical.

Suitably, the active component is homogenously dispersed within thepolymer matrix.

In a preferred embodiment, at least 90% of the coated microbeads in thepreparation have a diameter of from 200 microns to 300 microns.

In a preferred embodiment of the invention, the active agent is a cell,preferably a probiotic cell preparation.

The invention also relates to a dairy product comprising a preparationof coated microbeads according to the invention.

The invention also relates to a vehicle capable of delivering an activeagent to a lower intestine of a subject while protecting the activeagent during passage through the stomach of the subject, the vehiclecomprising a coated microbead preparation according to the invention.

The invention also relates to a method of delivering (i.e. releasing) anactive agent to a distal intestinal region of a subject and protectingthe active agent during passage through a stomach and proximalintestinal region of the subject, and in which the active agent issusceptible to degradation in the acidic conditions of the stomach, themethod comprising a step of orally administering to the subject apreparation of coated microbeads according to the invention. Typically,the active agent is a cell, ideally a probiotic bacterial preparation.

As indicated above, the term whey protein should generally be understoodas meaning a whey protein fraction having at least 30% β-lacoglobulin,for example various types of whey protein concentrates. Ideally, thewhey protein is a whey protein isolate (WPI) suitably having at least70% or 80% beta-lactoglobulin. The active component for example maybe acomponent that is sensitive to processing conditions, in-vivoconditions, or storage conditions, for example, probiotic bacteria whichare sensitive to damage by acidic gastric-transit conditions, or by highsheer or high pressure forces being exerted during processing. Thus, theactive component may be sensitive to pH, enzymes (i.e. proteaseenzymes), high pressure, high shear, and temperature abuse duringstorage. In one particularly preferred embodiment of the invention, theactive component is a cell, typically a bacterial cell, and ideally aprobiotic cell. Such cells are sensitive to low pH conditions, such aswould be encountered in the stomach, and as such need to be shieldedfrom gastric pH and bile salt environments. Probiotic bacteria, andindeed other types of cells, are also sensitive to high shear or highpressure, such as are employed in conventional methods of generatingmicron-sized polymer beads. Other types of active components which maybe encapsulated in the microbeads of the invention include enzymes,starter bacteria, cell extracts, proteins and polypeptides, sugars andsugar derivatives, nucleic acids and nucleic acid constructs,pharmaceutically-active agents, imaging dyes and ligands, antibodies andantibody fragments, pytochemicals and the like.

The invention also relates to a food product, especially a dairyproduct, comprising microbeads (coated or otherwise), or a preparationof microbeads (coated or otherwise), according to the invention.

The Applicant has surprisingly discovered that the microbeads of theinvention are capable of selectively removing phytochemical compoundsfrom liquids. As an example the Applicant has successfully removed alarge proportion of anthocyanin compounds from cranberry juice. Thesecompounds become part of the payload of the microbeads, and can besuccessfully delivered through the stomach and released in the smallintestine. The invention thus also relates to a method of removing aphytochemical fraction/product from a liquid comprising a step ofadmixing a preparation of microbeads according to the invention with theliquid to allow the microbeads adsorb a phytochemical fraction from theliquid. Examples of phytochemical compounds are polyphenolic compounds(i.e. anthocyanins), fat soluble vitamins, and the like.

Thus, the invention also relates to a preparation of bioactivemicrobeads comprising microbeads according to the present inventionhaving one or more phytochemical compounds adsorbed onto an outersurface of the microbeads. The method also relates to a method ofdelivering bioactive phytochemical compounds to the lower intestine of asubject comprising a step of orally administering a preparation ofbioactive microbeads to the subject. The invention also relates to amethod of making bioactive microbeads comprising a step of admixing apreparation of microbeads according to the invention with aphytochemical-containing fluid for a sufficient period of time to allowat least a portion of the phytochemical fraction of the fluid adsorb tothe microbeads and, optionally, removing the bioactive microbeads fromthe fluid. The invention also relates to a food product comprising apreparation of bioactive microbeads according to the invention. The foodproduct may be, for example, a dairy product such as a milk drink, ayoghurt or the like, or a fruit-based food or drink.

The invention also relates to a method of debittering a product (i.e.reducing the bitter taste of the product) for example a beverageproduct, for example a fruit-based beverage, of the type comprisingphytochemical bitter-tasting compounds, the method comprising a step ofadmixing a preparation of microbeads according to the invention with theproduct for a sufficient period of time to allow at least a portion ofthe phytochemical bitter-tasting compounds of the product adsorb to themicrobeads, and removing the microbeads and adsorbed bitter-tastingcompounds from the fluid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Inotech® Encapsulator and the flow process during the extrusionprocedure.

FIG. 2: Viability of probiotic cell population (Lb. rhamnosus GG) duringthe encapsulation procedure. 1=Fresh (19 hour) stationary phase cellsuspension, 2=Homogenized cell suspension, 3=protein-probiotic mixture,4=whey protein micro-beads, 5=Whey protein micro-bead+homogenization.Lb. rhamnosus GG yielded an extraction yield and encapsulationefficiency of 106.1±2% and 94.5±1.1%, respectively.

FIG. 3: The protein leakage expressed as protein loss (mg)/250 mL ofacetate buffer during micro-bead formation as a function of acetate pH.Protein concentration was determined according to the Bradford assay.

FIG. 4: Micro-bead strength as a function of curing time (0-180 minutes)in acetate buffer. Compression distance (♦) remains constant throughoutthe assay, indicating the presence of a monolayer micro-bead sample.

FIG. 5: Degree of hydrolysis of whey protein micro-beads as a functionof digestive media at 37° C. HCl pH 1.8 (♦), Intestinal juice pH 7.6 (▴)and phosphate buffered saline (PBS) pH 7.6 (●) were incubated for 180minutes at 200 rpm. Degree of hydrolysis was determined according to theOPA assay.

FIG. 6: Light microscope images of whey protein micro-beads far from theisoelectric point (pI) of whey proteins (A), close to pI (B). FIGS. 6 cand 6 d illustrate bead homogeneity and probiotic cell distribution,respectively.

FIG. 7: Size Distribution of whey protein micro-beads as a function ofcuring time at an acetate pH close to the pI of WPI as measured by laserdiffractometry and light microsopy. D (v, 0.9) represents 90% ifmicro-beads with a diameter below 282 μm and D (v, 0.5) represents 50%if micro-beads with a diameter below 212 um; however, light microscopymeasured an average diameter of 202.21 μm±0.04 μm.

FIG. 8: Particle size distribution of whey protein micro-beads at anacetate pH close to the pI of WPI (average size, 216.36±1.48 μm; size atpeak 201.68±0.43 μm) using laser diffractometry.

FIG. 9: Effect of microencapsulation on the survival of Lb. rhamnosus GGin ex vivo porcine gastric contents pH 2.0 at 37° C. ▴=Micro-beads pH2.0; ⋄=positive protein control pH 2; ▪=Free cells pH 2.0.

FIG. 10: Release of micro-encapsulated Lb. rhamnosus GG from wheyprotein micro-beads in ex vivo porcine gastro-intestinal contents at 37°C. ⋄=Micro-bead in gastric juice pH 2; ▴=Micro-beads in gastric juice pH2.4; ▪=Micro-beads in small intestinal juice (jejunum) pH 6.6.

FIG. 11: Visualisation of cell viability in a) small intestinal juicefollowing release of Lb. rhamnosus GG from whey protein micro-beadsafter gastric incubation (pH 2.0; 37° C.) and b) free cell control after15 min and c) 3 h gastric incubation (pH 2.0; 37° C.). These flowcytometry dot plots illustrate live, injured and dead cells in quadrantA3, A2 and A1, respectively.

FIG. 12: Size exclusion chromatogram illustrating the gradualdisintegration of whey protein micro-beads during incubation (0-180minutes) in porcine intestinal contents pH 6.6 at 37° C. (150 rpm).

FIG. 13: Zeta potential of whey protein micro-beads mixture (uncoated=1)coated with anionic polysaccharides (2=apple pectin, 4=citrus pectin,6=sodium alginate, 8=kappa carrageenan, 10=iota-carrageenan) andsubsequently coated (double coating) with 1% (w/v) heat-treated WPI, pH2.9 (3, 5, 7, 9 and 11). Error bars represent standard deviation of 3independent tests performed in triplicate.

FIG. 14: Microscope images clearly differentiating between themicro-bead matrix and the polysaccharide coating layer.

FIG. 15: Micro-bead diameter as a function of polysaccharide coating bymeans of layer-by-layer deposition.

FIG. 16: Storage survival of coated and non-coated micro-beads during14-day storage at ambient temperature storage in cranberry juice (pH2.6).

FIG. 17: Storage survival of coated and non-coated micro-beads during14-day storage during refrigerated storage (4° C.) in cranberry juice(pH 2.6).

FIG. 18: Storage survival of coated and non-coated micro-beads incranberry juice (refrigeration temperature 4° C.) followed by ex vivogastric incubation ph 1.8.

FIG. 19: Cell release from micro-beads during ex vivo intestinalincubation.

FIG. 20: Transit time of whey protein micro-beads as a function GIsection 2-(

) and 4-h (

) post probiotic administration. Data represents the mean values from 3pigs (n=3 per treatment) with standard deviation of the means indicatedby vertical bars. Asterisks denote significant differences (*p<0.05; ***p<0.001) in colony forming units 2 h post-probiotic administrationrelative to 4 h transition.

FIG. 21: Illustrates of the sequential evolution of LGG^(Rif) survivalwithin stomach (

), jejunum (

), ileum (

) and caecum (

) post-probiotic administration as a function of treatment group.

FIG. 22: Cell adhesion of LGG^(Rif) (

) and total Lactobacillus (

) to ileal tissue obtained 1 m proximal of the ileo-caecal junction. Thedata represents the mean of value of two animals per treatment.

FIG. 23: Absorption at 520 nm of grape, pomegranate and cranberry juicein the presence of whey protein micro-beads at 4 degrees and storage forup to 13 days.

DETAILED DESCRIPTION OF THE INVENTION Methods Chemicals

BiPro, a commercial whey protein isolate (WPI) was obtained from DaviscoFoods International Inc., (Le Sueur, Minn., U.S.A.). WPI nativeβ-lactoglobulin (β-Lg) and α-lactalbumin (α-La) content were analyzed byreverse phase-HPLC and estimated at 82% and 16%, respectively. Sodiumacetate was obtained from Sigma Chemicals Co. (Basingstoke, Hampshire,UK). Tween-20 and acetic acid were obtained from BDH (Alchem ChemicalsLtd., Little Island, Co. Cork, Ireland). The chemical products used inhigh performance liquid chromatography (HPLC) were acetonitrile (ACN)and trifluoroacetic acid (TFA), both HPLC grade. Highly purified water(Milli-Q PLUS, Millipore Corporation) was used in all cases fordispersion of samples, culture mediums and buffer solutions.

Probiotic Cultures & Cell Suspensions

In-Vitro and Ex-Vivo Data

The probiotic strain Lactobacillus rhamnosus GG (ATCC 53103, L.rhamnosus GG, Valio Ltd., Finland), was procured from University CollegeCork, under a restricted materials transfer agreement. Harvested cellswere stored as stock solutions in de Man Rogosa Sharpe (MRS) broth(Oxoid Ltd., Hampshire, U.K.) (REFERENCE: De Man, J. C., Rogosa, M. andSharpe, M. E. 1960. A medium for cultivation of lactobacilli. Journal ofApplied Bacteriology 23:130-135. containing 50% (v/v) aqueous glycerolat −20° C. All tests were performed using subcultures from the samefrozen stock, which was routinely checked for purity. Prior to assay, L.rhamnosus GG was serially transferred three times in MRS broth (OxoidLtd., Hampshire, U.K.) and incubated anaerobically at 37° C. for 24 hour(Merck, Darmstadt, Germany). Bacteria destined for encapsulation werepropagated from 1% (v/v) inoculums for 19 hour at 37° C. under anaerobicconditions. The probiotic biomass in early stationary phase (10⁹ cfu/mL)was harvested by centrifugation at 5,200×g for 10 minutes at 4° C.(Sorvall, RC-5C Plus, Sorvall Products, Stevenage, Herts, UK), washed,filter-sterilized and resuspended in sterile phosphate-buffered saline(Sigma Chemical CO, St Louis, U.S.A.). Fresh cells suspensions preparedfor each experiment were enumerated by pour plating in MRS agar(anaerobic incubation at 37° C. for 48 hour) and subsequently usedeither directly in the assay (free-cell condition) or employed withinthe microencapsulation process.

Microbead Matrix & Curing Media

WPI was hydrated in sterile water (11% w/v) for 16 h at 4° C. underslight agitation (180 rpm) in order to permit good protein hydration.WPI solution was adjusted to pH 7 with 100 mM HCl and filtered through0.45 μm (Millex, HVLP, Millipore Corp., Bedford, Mass.) to remove tracesof undissolved material. The protein dispersion was subsequently heatedat 78-80° C. for 30-45 minutes under agitation (to promote proteinpolymerization. The suspension of reactive WPI aggregates wassubsequently cooled on ice, stored at 4° C. overnight.

The curing media was formulated using an acetate buffer (0.5 M) adjustedto pH 4.6, where protein leakage was at a minimum value (see FIG. 3).This Following pH equilibration, 0.04-0.08% Tween-20 was added and thesolution was subsequently filtered (0.22 μm), sterilized (121° C. for 15minutes) and tempered at 35-37° C. prior to encapsulation experiment.

Production of Microbeads

Monodisperse whey protein micro-beads were prepared using anencapsulation device (Inotech Encapsulator® Inotech AG, Dottikon,Swtizerland) illustrated in FIG. 1. The technology is based on laminarjet break-up induced by applying a sinusoidal frequency with definedamplitude to a nozzle (3). The protein-probiotic suspension is deliveredto the nozzle via a feed line (1), which is connected to the polymerreservoir. Nozzles with diameters in the range 50-1,000 μm may be used.The nozzle is connected via a PTFE membrane, to a vibrating device (2),which is insulated from the surrounding structures by rubber mounts toavoid the generation of resonance frequencies in the system. The flow ofpolymer solution to the nozzle is accomplished using a precision syringepump with maximum extrusion volume of 50 mL.

The production of <50 mL of micro-beads was sufficient to meet therequirements of this study; hence the encapsulator was utilized as abatch-reactor. All glassware and solutions used in the procedure weresterilized (121° C. for 15 minutes) and the protein suspension wasequilibrated to room temperature, filtered sequentially through sterile5-, 1.2-, 0.8- and 0.45-μm pore sized filters (Sartorious) and mixedthoroughly with the bacterial concentrate to yield a probioticpopulation corresponding to the stationary phase concentration (10⁹CFU/mL). The protein-probiotic blend was aseptically extruded through a150-μm nozzle and subsequently passed through an electrode (4) into theencapsulation vessel (8) containing 250 mL of tempered (35° C.) curingbuffer solution (7), with continuous agitation to avoid coalescence ofmicro-beads during polymerization. A collection cup, suspended (5) fromthe top plate, was utilized during the initial priming of the nozzlewith protein-probiotic mixture. This facilitated the retrieval ofinitial polymer droplets with a diameter in excess of the predictedvalue (defined under controlled conditions) and thus ensuredmonodispersity of the subsequent micro-bead batch. The pliablemicro-beads formed were cured for 3 hour at room temperature in order toguarantee complete protein polymerization. The resulting rigidmicro-beads were recovered, washed twice in sterile water andsubsequently analyzed.

The flow rate of the protein-probiotic suspension, vibrational frequencyand vibrational amplitude were controlled as desired. The frequency maybe estimated from knowledge of the physiochemical properties of theprotein mixture for the chosen nozzle diameter; however in practice someadjustments were required to obtain a favorable micro-bead diameter.Consequently, the polymer mixture was delivered at a set feed rate tothe nozzle to achieve a steady i) jet of liquid and ii) flow rate (3-10mL/min), visualized with the aid of a stroboscopic device (6). Anoptimum vibrational frequency (900-3000 Hz) and amplitude (4.7 to 7)were defined thereafter to facilitate jet break-up and dropletproduction. Once these parameters were determined for a givenprotein-probiotic suspension, they were logged and utilized withoutadjustment for further batch production using identical polymerparameters.

Production of Polysaccharide Coated Microbeads

The following biopolymers were used as coating materials: Apple Pectin,Citrus Pectin, Sodium Alginate, Kappa carrageenan, iota carrageenan, andgum acacia. Pectin solutions were prepared in 10 mM phosphate buffer pH7 while the remaining biopolymers were solubilized in distilled water.After complete dissolution, the pH was adjusted to 4.6. All treatmentmaterials were filtered through 0.22 μm sterile filters. Each micro-beadpreparation (prepared as above) was cured for max. 3 h, washed twice insterile water and resuspended in 100 mL of the respective polysaccharidesolution (pH 4.6). Micro-bead batches were agitated (100 rpm) overnightat room temperature and subsequently cured for 15 min in 0.2M CaCl₂.Double coating: Additional batches from each biopolymer treatment werewashed and resuspended in heat-treated WPI (1% w/v: pH 2.9) and agitatedunder similar conditions.

Analytical Detection of Polysaccharide Coating

The electrophoretic mobility (EM) of coated whey protein micro-beads wasdetermined using a Zetasizer (Malvern, Worcs., UK) following ahomogenization (setting 3; 5 min) and centrifugation (5000×g; 5 min; 20°C.) procedure. EM was derived from the velocity of theprotein-polysaccharide suspension under an applied electric field of 150V and converted into zeta potential using the Helmholtz-Smoluchowskiequation.

In-Vivo Data

The probiotic strain Lactobacillus rhamnosus GG (ATCC 53103, Lb.rhamnosus GG, Valio Ltd., Finland), was procured from University CollegeCork, under a restricted materials transfer agreement and harvestedcells were stored as stock solutions in de Man Rogosa Sharpe (MRS) broth(Oxoid Ltd., Hampshire, U.K.) containing 50% (v/v) aqueous glycerol at−20° C. All tests were performed using subcultures from the same frozenstock, which was routinely checked for purity. The frozen culture wasgrown in MRS broth (Oxoid Ltd., Hampshire, U.K) at 37° C. underanaerobic conditions, achieved by activation of Anaerocult gas packs(Merck, Darmstadt, Germany). A spontaneous rifampicin-resistant(LGG^(Rif)) derivative, required to facilitate subsequent enumerationduring the pig trial, was isolated by spread plating 10⁹ cfu's from anovernight culture onto MRS agar containing 100 μg of rifampicin/ml(MRS^(Rif)) (Sigma Chemical Co., Dorset, U.K). Following anaerobicincubation at 37° C. for 48 h, single colonies were selected and stockedin MRS broth containing 40% (v/v) glycerol. Pulsed-field gelelectrophoresis was performed to ensure homology between band patternsof parent and variant strains of Lb. rhamnosus GG. Growthcharacteristics, heat and acid tolerance of both strains were alsoelucidated for further confirmation of strain similarity (data notshown).

Treatment Preparation (for In-Vivo Examples)

Prior to assay, LGG^(Rif) was serially transferred three times in MRSbroth (Oxoid Ltd., Hampshire, U.K.) and incubated anaerobically at 37°C. for 24 h (Merck, Darmstadt, Germany). Bacteria destined forencapsulation were propagated from 1% (v/v) inoculums for 19 h at 37° C.under anaerobic conditions. The probiotic biomass in early stationaryphase (10⁹ cfu/ml) was harvested by centrifugation at 5,200×g for 10 minat 4° C., washed, filter-sterilized and resuspended in sterilephosphate-buffered saline (Sigma Chemical Co., Dorset, U.K.). Freshcells suspensions were homogenized and enumerated on MRS agar asdescribed previously and subsequently blended with denatured wheyprotein to achieve a stationary-phase probiotic concentration (10⁹cfu/ml). This protein-probiotic blend was aseptically extruded through a150-μm nozzle for large-scale production of micro-beads as describedabove. Micro-bead batches containing 1.7×10¹⁰ cfu's, were polymerised,recovered and stored at 4° C. prior to animal dosing. Furthermore,coated micro-beads were prepared as described below by electrostaticdeposition of apple pectin (Cybercolloids, Cork, Ireland) onto thesurface of micro-beads following 2 h polymerization. A positive controlwas also prepared using a native (substrate) protein solutionaseptically blended with the cell concentrate in order to attainconsistent bacterial concentrations (10⁹ cfu/ml) among all treatmentsamples. Bacterial populations were determined by homogenisation,dilution and pour-plating on MRS agar.

Determination of Probiotic Viability During Cranberry Juice Storage andSubsequent Gastric Incubation

The viability and sensitivity of the encapsulated bacteria was evaluatedwithin various coated micro-bead environments, by storing all treatmentsat room temperature and refrigerated conditions for 28 days. Theenumeration of viable cells was conducted on days 0, 1, 4, 8 and 14 ofstorage. Entrapped bacteria were released from their respective proteinnetwork using a homogenization procedure (data not shown) to ensurecomplete liberation of bacteria from the respective protein systems.Planktonic Lb. rhamnosus GG cells (control) were treated similarly, tomaintain consistent treatment conditions. Homogenates were spread-platedon MRS agar and colonies were subsequently counted after 48 h incubationat 37° C. under anaerobic conditions. Plates containing 30-300 colonieswere enumerated and recorded as cfu/ml of protein material.

Probiotic Detection Using Flow Cytometry

In addition to plate counts, the viability of probiotic cells wasassessed by flow cytometry (FACS), using the BD Cell Viability assay (BDBiosciences, Oxford, U.K.). Digesta homogenates were diluted to apre-determined cell density and enzymatic treatment was performed inassociation with fluorescent staining. Data acquisition was performed ona BD FACS Canto II flow cytometer (BD Biosciences, Oxford, U.K.),equipped with 488 nm laser excitation and BD FACS Diva software using aside scatter (SSC) threshold.

Microscopy

Microscopy work was performed using a BX51 epifluorescence microscopeand a Leica TCS SP5 confocal scanning laser microscope (CSLM) (LeicaMicrosystems, Wetzler, Germany) as described by Doherty et al.(2010).Briefly, gastro-intestinal contents were stained by integrating the dyeconcentrations previously optimized during FACS analysis and imagedusing ×63 magnification objective with a numerical aperture of 1.4.Fluorescent and bright-field light microscopy were also performed onselected digesta sections using a BX51 light microscope (Olympus,Germany).

In-Vivo Transit Time Studies

Transit time of probiotic loaded micro-beads and sequential evolution ofLGG^(Rif) along the gastro-intestinal (GI) tract were investigated invivo due to there consideration as fundamental pre-requisites forsuccessful engineering of a full-scale animal trial. Feeding studieswere performed with pigs, in compliance with European Union CouncilDirective 91/630/EEC (outlines minimum standards for the protection ofpigs) and European Union Council Directive 98/58/EC (concerns theprotection of animals kept for farming purposes) and were approved by,and a license obtained from, the Irish Department of Health andChildren. Briefly, two weeks-post weaning, six male pigs were blocked byweight (mean weight of 13.2±0.6 kg), penned individually (as describedbelow) and randomly assigned to two groups (n=3). Having fasted from theprevious evening (16 h), both groups were dosed with proteinmicro-beads, loaded with 10¹⁰ cfu LGG^(Rif), delivered by means of ahighly palatable milk permeate (non-protein milk NPM) medium. Animalsreceived 200 ml NPM post-probiotic administration. Two (n=3) and 3 h(n=3) later, animals were sacrificed by captive bolt stunning followedby exsanguination, in the same order as they were fed. Previous markertransit studies in pigs showed that the majority of ingested feed wouldhave transited to the small intestine within 2 h; however sequentialrecovery of encapsulated probiotics may supersede these expectations dueto the nature of the delivery system. Hence, data relating to theevolution of LGG^(Rif) in the GI tract was elucidated by examiningstomach and ileostomy contents of all treatment animals andmicrobiological analysis is documented below.

In-Vivo Pig-Feeding Trial

A total of 32 (male) pigs from different litters of a conventionalcrossbred program (Large White×Landrace) performed in Moorepark PigProduction Development Unit (MPDU), were weaned at c. 26 days of age. At14 days post-weaning, (day −7) pigs were tagged, blocked by mean initialbody weight (11.8±1.3 kg) and randomly assigned to one of four treatmentgroups (n=8): control, probiotic substrate suspension, probioticmicro-beads and coated micro-beads. Pigs were housed individually inpens designed to provide reasonable space for free movement and normalactivity of pigs, thereby assuring normal GI motility (Snoeck et al.,2004). All pens, equipped with a single feeder and nipple drinker, werelocated in light-controlled (0700 h to 1630 h) rooms with temperaturesmaintained at 28-30° C. throughout the trial using a thermostaticallycontrolled space heater. The next 7 days were documented as theacclimatisation period (day −7 to day 0), during which animals were feda non-medicated commercial diet (free of antimicrobials, performanceenhancers, and sweeteners) twice daily at 0730 and 1530 (350 g/serving)with ad libitum access to fresh water. During this period, animals weretrained to consume a standard volume of carrier medium—pasteurized applejuice (Sgeez®, Dublin, Ireland)—within a defined time preceding theirmorning feed. Juice consumption, attitude of the animal and fecalconsistency were monitored daily during the acclimatization period andwere combined to generate a scoring system (data not shown). Any animalsshowing signs of ill-health were treated appropriately by on-sitepersonnel. Animals had unrestricted access to water at all times duringthe study.

On day 0, fecal samples were taken 6 h after the morning feed fordetermination of baseline parameters. Freshly voided feces (5-10 g) werecollected in sterile containers following rectal stimulation from twoanimals per treatment (n=8). Fecal samples were stored anaerobically at4° C. and microbiological analysis was performed within 2 h as outlinedbelow. In addition to the morning juice on day 0, all treatment groupswere offered an extra juice serving as a replacement for the eveningfeed. Following an overnight fast (16 h), animals received probiotictreatments with their morning juice on day 1. The first group (A)received free LGG^(Rif), which served as a control and the second group(B) was treated with native protein suspensions of LGG^(Rif).Probiotic-loaded micro-beads were administered to the third group (C),while the remaining group (D) received polysaccharide coated micro-beadsand no significant size difference existed between respective micro-beadtreatments. A dosing method was optimized to mitigate animal stressduring treatment administration, since animal handling retards GItransit time (Snoeck, et al., 2004). During probiotic administration,each pig received approx. 400 ml of apple juice containing c. 4.2×10⁷cfu/ml of LGG^(Rif) in respective treatments, which provided a totaldose of 1.7×10¹⁰ cfu/animal. Use of a single carrier medium and standardmicro-bead size are fundamental pre-requisites since fluctuating gastrictransit times may result from variation in carrier viscosity andmicro-bead size/density (Davis et al., 2001, Mpassi et al., 2001).Preliminary experiments (data not shown) revealed an encapsulationefficiency of 96.1%±0.7% for probiotic loaded micro-beads; thus controlsamples (group A and B) were adjusted to procure approximately equalLGG^(Rif) concentrations among all treatment groups.

Two hours post-probiotic administration animals from treatment groups A,B and C were sacrificed (as described previously) while group D wasrandomized between two transit times (2 and 3 h) for the investigationof delayed cell release from coated systems. The entire GI tract wasremoved from each carcass immediately after slaughter and digesta (5-10g) from various GI regions were aseptically collected. Gastric (pyloric)contents were sampled, after which intestinal digesta from a 1 m lengthof the jejunum was collected, beginning at a point 1 m distal from thepyloric valve. Digesta was also taken from a 1 m length of the ileum,starting at 15 cm proximal of the ileo-caecal junction, which wassequentially followed by collection of entire caecum contents. Allsamples were stored anaerobically and transported on ice to thelaboratory where microbiological analysis was performed within 8 h ofslaughter, as outlined below. Tissue samples were also collected duringcarcass dissection from stomach, jejunum and ileal regions describedabove. Small tissue specimens (1-2 cm) were immediately stored on icefor microscope and probiotic adhesion analysis; the latter beingperformed within 2 h of slaughter.

In-Vivo—Microbiological Analysis of Porcine GI Contents

Faecal samples were homogenised in Maximum Recovery Diluent (MRD) as10-fold dilutions (w/w) using a stomacher (Lab-Blender 400; SewardMedical, London, U.K.); however all GI digesta were serially dilutedusing phosphate buffer (pH 7; 0.5 M) and homogenised in an ice bath tofacilitate permanent inactivation of digestive enzymes and completeliberation of encapsulated bacteria. Appropriate dilutions werespread-plated on three media as described by Gardiner et al. (2004), forenumeration of LGG^(Rif), total lactobacilli and Enterobacteriaceae.Briefly, probiotic counts for Lb. rhamnosus GG were obtained using MRSagar containing rifampicin (Sigma Chemical Co., Dorset, U.K.) as aselective agent and 50 U/mL of nystatin (Sigma Chemical Co., Dorset,U.K.) to inhibit yeasts and moulds after anaerobic incubation for 2 daysat 37° C. (MRS^(Rif)). Fecal bacteria in the family ofEnterobacteriaceae were enumerated on violet red bile glucose agar(Merck, Darmstadt, Germany) incubated at 27° C. for 24 h (VRBA), whiletotal Lactobacillus counts were detected on Lactobacillus-selective agar(Rogosa et al., 1951) (Becton Dickinson, Cockeydville, Md.) followinganaerobic incubation for 5 days (LBS). LGG^(Rif) was easily identifiableon MRS^(Rif) due to its distinct colony appearance i.e. well-developed,round, milky colonies however, randomly amplified polymorphic DNA PCR(RAPD-PCR) was performed (detailed below) to validate the presence ofLb. rhamnosus GG on MRS^(Rif) plates. GI contents were analysed for pHdetermination (Mettler Toledo MP220 pH meter) within 6 h of slaughter.

In-Vivo—Adhesion Assay

Ileal tissue samples were rinsed gently in MRD to remove any looselyadhering digesta and were further washed by immersion in MRD andvigorous shaking for 60 s. Tissue samples were then homogenised in freshMRD as 10-fold dilutions using a stomacher (Lab-Blender 400; SewardMedical, London, U.K.). The resultant homogenate was further diluted10-fold in MRD and appropriate dilutions were plated on the three mediadetailed above for enumeration of adherent bacteria.

Anthocyanin Absorption by Microbeads in Fruit Juices

3.5 mg of micro-beads, prepared as described above, were mixed with 35mL of commercial grape, pomegranate and cranberry juice in sterile 50 mLplastic tubes and stored horizontally in the dark for up to 13 days.Samples were taken every 24 hours. For sampling, the microbeads wereallowed to settle for a minute before 2 mL of the juice sample wastaken, centrifuged at 20,000×g for 5 minutes, its absorbance at 520 nm(indicative of anthocyanins) was measured and returned back to the stocksample of juice. Additionally the sample was scanned from 350 to 720 todetect any possible change in the absorption spectra. The pH was alsoverified prior each measurement.

Results

The data clearly indicated that microbeads according to the inventionare capable of surviving passage through the stomach and rupturing todeliver the active agent in the lower intestine (see for example FIGS. 9to 12). Ex-vivo data additionally indicates that coated microbeads arecapable of delayed delivery to the small intestine, thus allowing distalintestinal delivery (i.e. delivery of the active agent in the ileum)(see for example FIG. 19). In-vivo data shows that the active agent, inthis case probiotic bacteria, retain their functionality and viabilityupon delivery in the intestine. Referring particularly to FIG. 22, bothuncoated and coated microbeads retain their ability to adhere to theileal mucosa, that adhesion is improved by coating of the microbeads,and that adhesion persists for at least 3 hours.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in construction and detail without departing fromthe spirit of the invention.

The invention claimed is:
 1. A method for producing microbeadscomprising an active component encapsulated within a gelled whey proteinmatrix, the method comprising the steps of providing a suspension ofdenatured whey protein and an active component, spraying the suspensionthrough a vibrating nozzle to generate microbeads having an averagediameter of from 80 to 500 μm, and immediately curing the microbeads byacidification by immersion in an acidic curing solution resulting ininstantaneous gelation upon contact with the acidic curing solution, andwherein the denatured whey protein comprises denatured β-lactoglobulin.2. A method as claimed in claim 1, wherein at least 90% of themicrobeads have a diameter ranging from 80 to 500 μm.
 3. A method asclaimed in claim 1 in which the pH of the acidic curing solution is from4.0 to 4.9.
 4. A method as claimed in claim 1 in which the whey proteinis a whey protein isolate.
 5. A method as claimed in claim 1 in whichthe whey protein has a degree of denaturation of at least 90% w/w.
 6. Amethod as claimed in claim 1 in which the whey protein suspension has aconcentration of 5-15% w/w whey protein.
 7. A method as claimed in claim1 in which the acidic curing solution has an acid concentration of from0.3M to 0.6M.
 8. A method as claimed in claim 1 in which the temperatureof the acidic solution is from 30° C. to 40° C.
 9. A method as claimedin claim 1 in which the acidic curing solution comprises a surfactant toprevent or inhibit agglomeration of the microbeads.
 10. A method asclaimed in claim 1 in which the microbeads are subject to an extendedcuring period in the acidic curing solution, for a period of at least 15minutes after gelation.
 11. A method as claimed in claim 2 in which thewhey protein has a β-lactoglobulin content of at least 30% w/w.
 12. Amethod as claimed in claim 1 in which the whey protein suspension issprayed through a vibrating nozzle and laminar break-up of the sprayedjet is induced by applying a sinusoidal frequency with defined amplitudeto the nozzle.
 13. A method as claimed in claim 1 in which the activecomponent is a suspension of cells.
 14. A method for producing coatedmicrobeads comprising the steps of producing microbeads according to amethod of claim 8, and resuspending the microbeads in a solution ofanionic polysaccharide having a pH of 4.6 or less for a period of atleast 60 minutes.